Methane-oxidizing bacteria (methanotrophs) are capable of growth on methane as their sole carbon and energy source. They are ubiquitous in the natural environment, and apparently play a significant role in carbon cycling in specific habitats. In addition to their ability to utilize methane, under laboratory conditions the enzyme catalyzing the oxidation of methane (methane monooxygenase; MMO) also cooxidizes a variety of non-growth substrates including straight-chain and cyclic hydrocarbons, as well as halogenated hydrocarbons. Since many of these compounds are toxic, it is of considerable interest to study this cooxidation phenomenon. However, some key aspects of this system are not well-understood. Depending on the growth conditions two different MMO's have been observed, one soluble and the other associated with the cell membrane. The membrane-associated MMO has a comparatively narrow substrate range, while the soluble enzyme has a much broader substrate range. Although the membrane-associated MMO appears to be the predominant activity under most growth conditions, it has not been purified and little information is available concerning its biochemistry, genetics or regulation. The goal of this project is to carry out a major study of the membrane- associated MMO in methanotrophs in order to assess the significance and potential of methanotrophs for detoxification of toxic hydrocarbons. We propose to use a dual approach to this problem. First, we will biochemically characterize genes for the membrane-associated MMO to assist in the biochemical studies, and to study regulation of this system. These studies will provide the information necessary to begin to understand the role of the membrane-associated MMO in methanotrophic physiology and in cooxidation processes.